EP0647716A1 - Vector comprising viral gene transcribed by ARN polymerase III - Google Patents

Abstract

The subject of the present invention is a recombinant vector comprising a cassette for transcription by RNA polymerase III, which cassette consists of a viral gene transcribed by RNA polymerase III into which an oligonucleotide DNA fragment has been inserted between or outside the boxes A and B constituting the said viral gene promoter. The subject of the present invention is also a process for the intracellular production of an RNA fragment in vitro or in vivo in which eukaryotic cells containing RNA polymerase III are transfected or infected with a vector according to the invention containing, as oligonucleotides, a DNA fragment corresponding to the reverse transcript of the said RNA and in that the eukaryotic cells thus transfected or infected are cultured in an appropriate culture medium. Finally, the present invention also relates to the use of the vectors as medicinal product.

Description

The present invention relates to recombinant DNA vectors, in particular of the plasmid or viral type, comprising a transcription cassette by RNA polymerase III constituted by a viral gene transcribed naturally by RNA polymerase III.

The present invention also relates to eukaryotic cells transfected or infected with vectors according to the invention.

The present invention also relates to a method of intracellular production of an RNA fragment in vitro or in vivo by culture of eukaryotic cells transfected or infected with the vectors according to the invention.

The present invention also relates to the use of vectors according to the invention as a medicament.

Antisense molecules are RNA sequences which selectively hybridize to messenger RNAs of which they are complementary and thus block the expression of the gene considered (maturation of RNA, translation). The work carried out since the early 1980s attests to the effectiveness of such a system for suppressing numerous cellular or viral functions (1,2).

The prospects for potential applications of these antisense molecules are important in basic research. The use of antisense to correct the expression of certain "abnormal" phenotypes or to fight effectively against viruses for which conventional approaches (vaccination, chemotherapy ...) prove to be delicate, as is the case for the virus of Human Immunodeficiency (HIV).

One of the problems posed by the use of antisense lies in the fact that these molecules are effective at significant intracellular concentrations much much higher than those of their substrate. The intracellular concentration of antisense molecules being the result of the difference between synthesis and degradation, solutions were sought according to the invention which favor the synthesis and the stability of the products formed. In this perspective, the operating efficiency of the antisense molecule vis-à-vis its target is an important parameter since the more a molecule is "powerful" the less it will require a high concentration to obtain a maximum effect.

RNA polymerase III is the eukaryotic enzyme responsible for the synthesis of a wide variety of small cytoplasmic or nuclear RNAs. The main representatives are transfer RNAs (tRNA) or ribosomal RNAs of type 5S (3).

RNA polymerase III is capable of efficiently transcribing cloned DNA fragments into the acellular system. This is possible provided that these DNA fragments contain the promoter regions specific for transcription by polymerase III. These promoter regions are now well characterized (4) and comes out above all constituted by intragenic regions arranged in a discontinuous manner. In the case of tRNA type genes, these are two regions of DNA: "box A" and "box B" each formed by II nucleotides (consensus sequence) and spaced by an intermediate region of variable size ( 4).

The RNA transcribed by polymerase III are remarkable for their small size and especially for their conformation in space. Thus, the clover leaf structure of the tRNAs is particularly well known. This secondary structure probably ensures the tRNA stability but above all functionality. The sequences present at the level of the loops (anticodon) are free to hybridize on a complementary RNA. Cotten and Birnstiel in 1989 (5) proposed to insert a "ribozyme / antisense" sequence in the tRNA ^met gene of Xenopus. The oligonucleotide was inserted into the intermediate region located between boxes A and box B of the promoter regions so as to be correctly presented at the level of the anticodon (ribtRNA ^met , reference 2).

Some viruses (Adenovirus, Epstein Barr virus, Herpes virus) have genes transcribed by RNA polymerase III. In particular, Adenoviruses have two genes, of similar organization, called VAI and VAII (VA: Virus Associated) coding for two different VA-RNA VA-RNAI and VA-RNAII (6). The VA genes are naturally cloned and functional in the genome of the Adenoviruses. There are more than 10 ⁷ VA-RNA molecules in a cell infected with an Adenovirus. These small RNAs, comprising 150 nucleotides, are transcribed from a promoter comprising two distinct regions (box A and box B) located in the L1 region of the Adenovirus genome (7). VA-RNA has an effect on translation: adenovirus mutants defective in VA-RNA normally express their messenger RNAs but are unable to translate them efficiently. The explanation for this phenomenon is that the DAI protein kinase induced by interferon is inhibited by VA-RNAI, which allows its eIF-2 substrate to escape phosphorylation and therefore inactivation (9) ( the factor eIF-2 being necessary for the initiation of translation). The interaction between VA-ARNI and kinase has been well documented (10,11). They described two distinct regions of RNA, one involved in the binding of RNA with the DAI protein (nucleotides 93 to 136) and the other in the inhibition of kinase activity (nucleotides 54 to 77).

Jennings and Molloy (12) have shown that it is possible to repress the expression of a replicon of the SV40 virus in COS1 cells, using an anti SV40 antisense of 163 bp (T antigen) grafted at the end. 3 'of the VAI gene. After transfection of the COS cells by various constructions (sense, antisense), the authors show on a transient expression system that the T antigen is repressed at 50%.

According to the present invention, a short antisense sequence has been inserted inside the VA gene, with the aim of obtaining a functional assembly, that is to say retaining the partially double-stranded structure of the VA gene and its conformation. loop, hairpin type, which interacts with protein kinase. This maintenance of the structural configuration results in greater intracellular stability, on the one hand and a conservation of the functionality of the VA gene, on the other hand.

It has indeed been found, according to the present invention, that the remarkable efficiency of the functioning of these small RNAs in their role of inhibiting the activity of the protein kinase DAI can be used by diverting it from its primary vocation and by directing to another target via antisense or ribozymes. According to the invention, these VA genes are therefore used as a shuttle system, in particular for the development of a new family of antisense RNA molecules expressed in eukaryotes and potentially usable for a wide range of applications.

We have, according to the present invention, developed a gene model "cassettes" allowing the easy insertion of antisense oligonucleotides or ribozymes in the VA gene without affecting the rate of transcription and we were able to measure the relative efficiency of these constructions in terms of inhibition of replication of the HIV virus in culture. The present invention is in fact based on the use of RNA Polymerase III to efficiently transcribe cloned genes (VAI) carrying antisense or ribozyme sequences (in the form of exogenous DNA fragments of small sizes -15 to 25 nucleotides- inserted in the gene under consideration).

In its most general aspect, the present invention relates to a recombinant DNA vector comprising a transcription cassette by RNA polymerase III, consisting of a viral gene transcribed by RNA polymerase III into which an oligonucleotide has been inserted. , DNA fragment, between or outside boxes A and B constituting said promoter of the viral gene.

The rate of transcription of VA genes by polymerase III is very important. This enzyme is well preserved in most eukaryotes and therefore adaptable to many cellular or animal models, the ubiquitous representation of this enzyme in all tissues does not pose any specific tissue limitation.

The use of these viral genes transcribed in large quantities, stable and functional, therefore makes them particularly efficient systems for the production of RNA in situ.

The organization of genes transcribed by this enzyme is interesting due to the absence of regulatory DNA sequences in the "extragenic" position, which simplifies the transcriptional regulatory scheme and ensures compacting of genetic information. The intragenic position of the promoters avoids having to manipulate non-transcribed regions, which makes it possible to manipulate small genes which are easy to clone. Finally, the viral genes according to the invention, such as VA, offer numerous possibilities for the insertion of oligonucleotides without affecting their rate of transcription and the secondary structure of the transcribed RNA.

In a suitable embodiment, the viral gene is a VA gene from an adenovirus, or an EBR gene from an Epstein Barr virus or a viral gene transcribed by RNA polymerase III from a Herpes virus.

In particular, mention may be made of the VAI or VAII gene of an Adenovirus, in particular the Adenovirus 2.

The VAI gene of the Adenoviruses was chosen as a pilot system, as an example of embodiment of the invention, because it is particularly well described in the literature. However, all of the VAI-like viral genes transcribed by RNA Polymerase III may also be suitable.

As a suitable vector according to the invention, there may be mentioned a plasmid or episomal replicative vector or a viral vector.

In particular, an episomal vector carrying the origin of replication of the Epstein Barr virus (oriP) will be used, as well as the sequences coding for the protein EBNA-1.

As regards the choice of the vector, it is in fact preferable to remain as close as possible to the "natural" system while disturbing it as little as possible. For this reason, it is preferable to use a vector which replicates autonomously (episome). The use of an episomal replicating vector may be a good means of expressing antisense in eukaryotic cells and in particular in the T lymphocyte. The cloning of the VA / antisense genes into vectors replicating in episomal form in the eukaryotic system is particularly suitable. . These vectors carry the origin of replication of the Epstein Barr virus (OriP) as well as coding sequences for the Ebnal protein strictly necessary for the replication of the DNA molecule carrying Ori P.

There is a greater operating efficiency of polymerase III on episomes as well as stimulation of transcriptional activity in the presence of the protein E1 a. These properties are used to improve the proposed system.

The viral vector according to the invention is preferably a DNA virus, but can also be a retroviral RNA vector; it will then include the RNA transcript of the transcription cassette according to the invention. Given the fact that infection of a cell by a retrovirus results in the production of circularized proviral DNA, it is this proviral DNA which will in turn be transcribed by RNA polymerase III according to the invention, this not excluding the functioning of the genetic construction used once the retroviral DNA is integrated.

In an appropriate embodiment according to the invention, the oligonucleotide is inserted into the intermediate regions located between the boxes A and B of the VA gene.

For a therapeutic application, preferably, the VA gene is inactivated as regards the inhibition of the action of interferon, by deletion or mutation in the VA gene of the elements of sequence critical for the inactivation of the protein kinase , DAI, induced by interferon.

This inactivation can be done by direct insertion of the oligonucleotide in the regions responsible for the activity of inhibiting the action of interferon, a region described to be in the VA 1 gene of Adenovirus 2 between and y including nucleotides 10672 and 10745. Also by this means, the affinity of the oligonucleotide for the target sequence is optimized, taking into account the spatial configuration of this loop-shaped region.

The integrity of the central region of the VA gene of nucleotides 10694 to 10730 (loop IV) is indeed critical for the maintenance of the inhibitory activity of VA RNA with respect to P68 kinase. This region is located outside the regulatory zones (boxes A and B) and its deletion does not affect the transcription of the VA gene.

According to a first embodiment of the present invention, the natural activity of the VA 1 gene of the Adenovirus 2 is inactivated by the insertion of the antisense in the central region of nucleotides 10694 to 10730, more particularly 10702 to 10728, or in place of this region which has been deleted.

However, in order to be able to construct chimeric VA-antisense genes for which the antisense is introduced outside this region, in another embodiment, a VA gene deleted from the central region (nucleotides 10702 to 10728) is constructed. In practice, the "overlap" PCR (FIG. 7) allows these deletions to be carried out when the oligonucleotides a 'and b' are spaced from the region to be deleted. In addition, a new restriction site (EcoRV; GATATC) can be created in particular by the juxtaposition of the two ends framing the deletion (GAT and ACC) thanks to the mutation of nucleotide 10729 (ACC replaced by ATC). This additional site can be used later for the cloning of antisense or ribozymic sequences in place of the deleted IV loop. This gene is called VA delta IV.

• - boucle I, nucléotides de 10635 à 10639 (-TAAAT-), située entre les boites A et B.
• - boucle III, nucléotides de 10682 à 10688 (-ATCCGGC-), située après la boite B.
• - boucle V, nucléotides de 10733 à 10736 (-GGTG-)

In addition to the newly created EcoRV site, other regions of the VA gene can serve as sites for the insertion of exogenous sequences (by PCR overlap), and in particular the regions corresponding to the single-stranded loops of the VA RNA. The insertion of exogenous antisense sequences into one of these regions or in place of one of these regions, the latter being deleted, allows the added oligonucleotides to appear on the secondary structure as "extrusions" with respect to the main axis of VA RNA. As a result, they are accessible for sense RNAs. We quote mainly, from 5 'to 3', the following single-strand regions:

• - loop I, nucleotides from 10635 to 10639 (-TAAAT-), located between boxes A and B.
• - loop III, nucleotides from 10682 to 10688 (-ATCCGGC-), located after box B.
• - loop V, nucleotides from 10733 to 10736 (-GGTG-)

Furthermore, the terminal end of the VA delta -IV gene can also be used as an insertion site. The added sequences are then placed just upstream of the stop sequence (-TTTT-) and define an extension with respect to the VA molecule. The unique Eco47111 site (-AGC / GCT) of nucleotides 10758 to 10763 can be used for this purpose.

In a particular embodiment, the oligonucleotide is inserted at the level of nucleotide 10711 of the VAI gene of the Adenovirus 2 represented in FIG. 1.

Preferably, the oligonucleotide according to the invention comprises from 15 to 40 nucleotides, more preferably still 15 to 25.

In the context of a therapeutic application, the oligonucleotide may correspond to an antisense RNA molecule or to a ribozymic RNA structure.

In order to improve the efficiency of hybridization between the antisense RNA molecule and its substrate, many studies are currently directed towards the search for target sequences correctly defined in terms of hybridization parameters (affinity, accessibility).

Ribozyme sequences (25) are minimum consensus sequences required for one RNA molecule to be able to hydrolyze another RNA molecule in a catalytic fashion. These catalytically active ribonucleotides (ribozymes) are capable of cleaving a target RNA on which they are specifically hybridized (thanks to two sequences of 15 nucleotides complementary to the target RNA and placed on either side of the catalytic sequence) . These ribozymic sequences comparable to "super antisense" can be used with profit in our system by increasing the functional efficiency of the whole.

• - additivité des gènes et donc augmentation de la concentration intracellulaire des ARN antisens.
• - multiplicité des séquences cibles et donc meilleure efficacité de fonctionnement.

Furthermore, the VA gene is small (160 nucleotides for VA 1 of the adenovirus 2). This allows according to the present invention to simultaneously use several VA-antisense genes carried by the same genetic construction. The aim is to define antisense "cocktails" (for example in the case of HIV concomitant use of three different genes; anti-rev, anti-tat and anti-packaging signal). These multiple genetic constructions have two important advantages:

• - additivity of genes and therefore increase of the intracellular concentration of antisense RNA.
• - multiplicity of target sequences and therefore better operating efficiency.

In addition, in the case of viruses endowed with a strong genetic variability and which "adapt" to the treatment used, the recourse to the use of several antisense sequences makes it possible to avoid the emergence of genetic variants.

The present invention therefore also relates to a vector comprising several identical or different viral genes transcribed by RNA polymerase III into which an identical or different oligonucleotide has been inserted outside the boxes A and B of each of said viral genes.

The present invention also relates to a process for the intracellular production of an RNA fragment in vitro or in vivo in which eukaryotic cells comprising RNA polymerase III are transfected or infected with a replicative vector according to the invention, comprising as an oligonucleotide a DNA fragment corresponding to the reverse transcript of said RNA and in that the eukaryotic cells thus transfected or infected are cultured in an appropriate culture medium.

The present invention also further relates, as we have seen, to the use, as a medicament, of a vector according to the invention, in which the oligonucleotide is transcribed into an RNA molecule "antisense" or a ribozymal RNA molecule blocking the expression of a gene involved in a pathology by hybridizing and, if necessary, by cutting its messenger RNA of viral, bacterial or parasitic cell origin.

The medicament according to the invention can be used as an antiviral, antitumor, antibiotic or antiparasitic agent, or in any pathology where a gene is abnormally expressed either by mutation or by deregulation.

The present invention also relates to a method for blocking the expression of a gene in vivo using a vector according to the invention, the said oligonucleotide of which is transcribed into an antisense or ribozymic RNA molecule which hybridizes to , or respectively, cuts the messenger RNA of said gene.

The present invention also relates to a method of treating cells ex vivo using a vector according to the invention.

In its in vivo or ex-vivo therapeutic applications, a viral or retroviral vector which penetrates into the cell by transfection or infection is entirely appropriate. In particular as a medicament according to the invention, a vector is used consisting of a defective viral vector such as an adenovirus or a defective retroviral vector such as a murine retrovirus.

In fact, the vector used to convey the gene construction according to the invention to its theoretical target can be a retroviral vector with transport of the recombinant construction by a borrowing capsid and insertion of the genetic material into the DNA of the host cell.

• - les cellules sanguines sont aisément prélevables sans traumatisme pour le patient. De plus, les conditions de culture de ces cellules (utilisation de diverses cytokines) se sont affinées et permettent un maintien ex vivo pour des périodes de temps variables (congélation, culture).
• - l'étude de la différenciation des différentes lignées composant le système hématopoïétique a permis de montrer que l'ensemble de ces lignées dérivent d'un progéniteur commun (Uchida N., Fleming W.H., Alpern E.J. and Weissman I.L. 1993 Current Opinion in Immunology, 5, 177-184). Cela permet d'introduire la modification génétique souhaitée dans des cellules souches qui, après réimplantation, sont capables de recoloniser en totalité le tissu hématopoïétique.

Techniques of using vectors, including viral vectors, to transport and penetrate genetic material into target cells and effectively introduce genetic changes in various somatic tissues such as muscle, liver, brain and hematopolitic cells are known to those skilled in the art. In particular, hematopoietic tissue (leukocytes, red blood cells, platelets, etc.) has two essential characteristics that make this tissue a good candidate for gene therapy approaches:

• - blood cells are easily removed without trauma to the patient. In addition, the culture conditions of these cells (use of various cytokines) have been refined and allow ex vivo maintenance for variable periods of time (freezing, culture).
• - the study of the differentiation of the different lines composing the hematopoietic system made it possible to show that all of these lines derive from a common progenitor (Uchida N., Fleming WH, Alpern EJ and Weissman IL 1993 Current Opinion in Immunology, 5, 177-184). This makes it possible to introduce the desired genetic modification into stem cells which, after reimplantation, are capable of completely recolonizing the hematopoietic tissue.

Characterization of the precursor cells has made it possible to establish that the presence of a surface protein (CD34) makes it possible to distinguish them from other cell types as being CD34 + cells.

At all stages of differentiation, hematopoietic cells proliferate. Therefore, the introduction of foreign genes into these cells requires that it is not "diluted" during successive cell divisions. Retroviruses, which integrate definitively into the genome of the recipient cell and for which the molecular mechanism of replication is relatively well known, appear to be good candidates for hematopoietic cell gene therapy.

• - Dans un premier temps, les techniques de génie génétique permettent de modifier le génome d'un rétrovirus murin comme le virus de Moloney (rétrovirus murin appartenant au groupe des virus des leucémies murines : Reddy E.P., Smith M. J. and Aaronson S.A., 1981, Science. 214, 445-450). Le génome rétroviral est cloné dans un vecteur plasmidique puis délété de l'ensemble des séquences virales codant pour les protéines de structure (gènes : Gag, Env) ainsi que la séquence codant pour les activités enzymatiques (gène : Pol). De ce fait, seules les séquences nécessaires "en cis" pour la réplication, la transcription et l'intégration sont conservées (séquences correspondants aux deux régions LTR, signal d'encapsidation et signal de fixation de l'amorce). Les séquences génétiques délétées peuvent être remplacées par des gènes non viraux comme le gène de résistance à la néomycine (antibiotique de séléction pour les cellules eucaryotes) et par le gène à transporter par le vecteur rétroviral.
• - Dans un second temps, la construction plasmidique ainsi obtenue est introduite par transfection dans les cellules d'encapsidation. Ces cellules expriment de manière constitutive les protéines virales Gag, Pol et Env, mais l'ARN codant pour ces protéines est dépourvu des signaux nécessaires à son encapsidation. De ce fait, cet ARN ne peut pas être encapsidé et permettre la formation de particules virales. Seul l'ARN recombinant, issu de la construction rétrovirale transfectée, est dotée du signal d'encapsidation et est encapsidée. Les particules rétrovirales produites par ce système contiennent l'ensemble des éléments nécessaires pour l'infection des cellules cibles (telles que les cellules CD34+) et pour l'intégration définitive du gène d'intérêt dans ces cellules. L'absence des gènes Gag, Pol, Env empêche le système de continuer à se propager.

The use of retroviral vectors to transport genetic material requires, on the one hand, to carry out the genetic construction of the recombinant retrovirus, and on the other hand, to have a cellular system which ensures the function of packaging of the genetic material. to carry :

• - Initially, genetic engineering techniques make it possible to modify the genome of a murine retrovirus such as Moloney virus (murine retrovirus belonging to the group of murine leukemia viruses: Reddy EP, Smith MJ and Aaronson SA, 1981, Science . 214, 445-450). The retroviral genome is cloned into a plasmid vector and then deleted from all of the viral sequences coding for the structural proteins (genes: Gag, Env) as well as the sequence coding for the enzymatic activities (gene: Pol). Therefore, only the sequences necessary "in cis" for replication, transcription and integration are preserved (sequences corresponding to the two LTR regions, packaging signal and primer binding signal). The deleted genetic sequences can be replaced by non-viral genes such as the neomycin resistance gene (antibiotic for selection for eukaryotic cells) and by the gene to be transported by the retroviral vector.
• - In a second step, the plasmid construction thus obtained is introduced by transfection into the packaging cells. These cells constitutively express the viral proteins Gag, Pol and Env, but the RNA coding for these proteins lacks the signals necessary for its packaging. Therefore, this RNA cannot be packaged and allow the formation of viral particles. Only the recombinant RNA, originating from the transfected retroviral construct, is endowed with the packaging signal and is packaged. The retroviral particles produced by this system contain all of the elements necessary for the infection of target cells (such as CD34 + cells) and for the definitive integration of the gene of interest into these cells. The absence of the Gag, Pol, Env genes prevents the system from continuing to spread.

According to the present invention, the VA-antisense genes have the characteristic of being transcribed by RNA Polymerase III. This feature led to the development of two types of retroviral constructs in which the VA-anti rev genes were cloned as described in Example 6.

DNA viruses, such as adenoviruses, may also be suitable for this approach, although in this case maintaining DNA in an episomal state as an autonomous replicon is the most likely situation.

It goes without saying that the use of a viral vector, originating from an Adenovirus, is particularly suitable when the viral gene is an Adenovirus gene. Adenoviruses have certain interesting properties. In particular, they have a fairly broad host spectrum, are capable of infecting quiescent cells, and they do not integrate into the genome of the infected cell. For these reasons, adenoviruses have already been used for gene transfer in vivo. To this end, different vectors derived from adenoviruses have been prepared, incorporating different genes (beta-gal, OTC, alpha-1At, cytokines, etc.). To limit the risks of multiplication and the formation of infectious particles in vivo, the adenoviruses used are generally modified so as to render them incapable of replication in the infected cell.

Thus, the adenoviruses used are generally deleted from the E1 regions (E1 a and / or E 1 b) and possibly E3.

The defective recombinant adenoviruses according to the invention can be prepared by any technique known to a person skilled in the art (Levrero et al., Gene 101 (1991) 195, EP 185 573; Graham, EMBO J.3 (1984) 2917). In particular, they can be prepared by homologous recombination between an adenovirus and a plasmid in an appropriate cell line. The cell line used must preferably (i) be transformable by said elements, and (ii), contain the sequences capable of complementing the part of the defective adenovirus genome, preferably in integrated form to avoid the risks of recombination. As an example of a line, mention may be made of the human embryonic kidney line 293 (Graham et al., J. Gen. Virol 36 (1977) 59) which contains in particular, integrated into its genome, the left part of the genome d '' Adenovirus Ad5 (12%).

Then the adenoviruses which have multiplied are recovered and purified according to conventional molecular biology techniques.

According to the present invention, in the genome of the defective recombinant adenovirus is inserted, at the level of the VA gene, an exogenous DNA sequence in particular coding for an antisense RNA.

Pharmaceutical compositions comprising one or more virus vectors such as defective recombinants as described above can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, etc. administration. Preferably, they contain pharmaceutically acceptable vehicles for an injectable formulation. They may in particular be saline solutions (monosodium phosphate, disodium, chloride of sodium, potassium, calcium or magnesium, etc., or mixtures of such salts) sterile, isotonic, or of dry composition, in particular lyophilized, which, by addition according to the case of sterilized water or physiological saline, allow the constitution of solutes particular injectables of sterile, isotonic solutions, or dry compositions, in particular lyophilized, which by addition, as the case may be, of sterilized water or physiological saline, allow the constitution of injectable solutes.

The doses of defective recombinant virus used for injection can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the pathology concerned, of the gene to be expressed, or even of the duration of the treatment sought. In general, the recombinant viruses according to the invention can be formulated and administered in the form of doses of between 10 and 10 pfu / ml, and preferably 10 to 10 pfu / ml. The term pfu ("plaque forming unit") corresponds to the infectious power of a virus solution, and is determined by infection of an appropriate cell culture, and measures, generally after 48 hours, the number of plaques of infected cells. The techniques for determining the pfu titer of a viral solution are well documented in the literature.

The use of genetically modified viruses as a shuttle system to transport the modified genetic material not only allows the genetic material to enter the recipient cell through the use of a borrowing viral capsid, but also makes it possible to treat simultaneously, and over a short period of time, a large number of cells; this opens the way to curative approaches aimed in vitro at cells already infected with the virus to be inhibited, but also allows the therapeutic treatment applied to the whole organism.

According to the invention, viral transactivators can be used. In particular, the protein E1 of Adenovirus stimulates the transcriptional activity of polymerase III (23), in particular by mobilizing the limiting factor TFIIIC. This potentiating effect of polymerase III is especially observed if the DNA carrying the VA gene is in episomal form (frequent case for Adenoviruses). This property of the E1A protein can be used to stimulate the efficiency of the "VA-RNA polymerase III" system, especially during transient expression experiments, either with the E1 gene cis-cloned by the VA-RNA gene, or in trans by cotransfection.

Also, the Tat protein of HIV could stimulate the transcriptional activity of polymerase III.

• - Dans le but de conférer une "immunité cellulaire" à des cellules permissives à l'infection HIV (CD4+), le traitement est réalisé "ex-vivo" par le prélèvement de cellules souches issues de la moelle, leur tri et leur modification génétique "ex-vivo " _;
• - Une stratégie "curative" est également possible grâce à l'instauration d'une dépendance de la réplication du vecteur pour la présence de la protéine Tat du HIV. Cette dépendance permet de répliquer efficacement le vecteur uniquement dans des cellules infectées par le virus.

The use of viral transactivators coupled with the vectorization of the antisense can be used according to the invention, in terms of specificity of action with respect to cells infected with HIV (or other pathogenic viruses). Two complementary strategies can be applied:

• - In order to confer "cellular immunity" to cells permissive to HIV infection (CD4 +), the treatment is carried out "ex-vivo" by the removal of stem cells from the marrow, their sorting and their genetic modification "ex-vivo"_;
• - A "curative" strategy is also possible thanks to the establishment of a dependence on vector replication for the presence of the Tat protein of HIV. This dependence makes it possible to efficiently replicate the vector only in cells infected with the virus.

The development and development of antisense cassettes according to the invention, adaptable to many research subjects are envisaged in very numerous fields including in particular molecular oncology, cellular determinism.

Other characteristics and advantages of the present invention will appear in the light of the detailed description of the following embodiment.

Figure 1 represents the nucleotide sequence of the VAI gene of Adenovirus 2.

CAPTION of Figure 1:

• Figure 2 shows antisense and random sequences inserted at nucleotide 10711 of the VAI gene.
• FIG. 3 represents the analysis on acrylamide / urea gel of the acellular transcripts of the PVV2 / VAI / antisense constructs.
• FIG. 4 represents the kinetics of HIV infection of EMFs transfected or not with an antisense.
• FIG. 5 represents the assay of the HIV antigens present in the supernatants of the CEM cells transfected with antisense constructs.

The assays for HIV antigens were carried out 5 hours after renewal of the culture supernatant and 24 days after the start of the infection.

Secondary structures of VA-RNA: the structure VA-RNAI (A) and two possible structures for VA-RNAI (B and C) are described in reference 13.

• FIG. 6 represents the secondary structure of the VA I gene.
• Figure 7 shows the diagram of insertion of an oligonucleotide by PCR method "overlap".
• Figure 8 shows the expression of VA-antisense in the AS ₁₀ population
• lane a: RNA of cells from the AS ₁₀ population
• lane b: RNA from cells of the HepG ₂ line infected with type 2 adenovirus.
• FIG. 9 presents Northern-Blot results of the RNAs transcribed from a plasmid carrying the VA-anti rev and VA anti-tat genes obtained in Example 5 with the construct carrying the rev gene and the tat gene.
• FIG. 10 represents the construction of the retroviral vectors 1 and II derived from the vector pMV7 of Example 6.
• FIG. 11 represents the transient expression of the VA 1 genes modified from Example 7 in 293 cells.

EXAMPLE 1 Cloning of the VAI Gene

The gene chosen for the experiments proposed below is the VA-RNAI gene of Adenovirus 2. The sequence of Adenovirus is available in the database: Genbank - ref .: ADBVAI sequences transcribed from nucleotide 10610 to nucleotide 10769 (Figure 1 shows the sequence elements involved). The conformation of the RNA in solution is published and presented in Figure 6 (13,14).

The cloning of the recombinant VAI gene is carried out in a plasmid vector of the PVV2 type (15) carrying in cis the gene for resistance to geneticin (under dependence of the TK promoter) or in a vector with autonomous replication in eukaryotic system of the pCEP-4 type. (marketed by Invitrogen) or pHEBo (kindly provided by RP SEKALY, laboratory of Immunology, Montreal). These latter vectors carry the OriP and Ebna-1 regions and they also carry the hygromycin resistance gene (16,17).

The Adenovirus 2 VAI gene was cloned after a PCR amplification step. The oligonucleotides used for this PCR were chosen on the 5 ′ side upstream of the transcription start site of the VAI gene and on the 3 ′ side downstream of the termination TTTT site (FIG. 1). The 5 ′ ends of each oligonucleotide are provided with the Clal enzymatic cleavage site, a unique site on the plasmid pVV2, or else with the Hindlll site for cloning on the plasmid pHEBo. The transformation of bacteria with "pVV2-VAI" constructs made it possible to obtain several recombinant clones.

Example 2: Insertion and cloning of the antisense sequences

For HIV, the sequence of oligonucleotides to be inserted was first determined in accordance with the antisense sequences already published by other authors: anti-rev (18) or anti-tat (19) and random sequence serving as specific control ( see Fig. 2) (tat and rev proteins are two viral proteins whose regulatory role is critical for virus replication).

The use of ribozyme-type sequences is currently in progress, with the cloning of recombinants carrying ribozyme sequences published by Goodchild & Kohli in 1991 (20).

The use of "overlap" PCR (see FIG. 7) has made it possible to insert oligonucleotides of variable size into the VAI gene. The "anti-rev", "anti-tat" and "random" antisense were inserted at nucleotide 10711 of the VA-RNAI sequence (FIG. 2). The VAI genes thus modified by the insertion of the antisense sequence are cloned into the ClaI site of the plasmid pVV2 as described above.

The constructions obtained were all sequenced (on 200 nucleotides) and then tested in a cell-free transcription system to ensure the functionality as well as the relative transcription efficiency of each construction. For the development of in vitro transcriptions, the protocol followed is that described by Wu (21) and Weil (22). Briefly, 3 μg of DNA to be tested are incubated for 90 min at 29 _° C. in the presence of cell extracts containing polymerase III activity and of nucleotides, in particular of alpha- ^p32- dGTP. After synthesis, the reaction products are analyzed on acrylamide gel (Figure 3 shows the type of results obtained). The sizes observed correspond well to the sizes expected for each construct, namely: native VA-ARNI 160 nucleotides, VA-RNAI / anti-rev 188, VA-RNAI / anti-state 190. The other VA-RNAI / ribozyme constructions are not not yet analyzed.

It is well verified that the insertion of exogenous sequences into the VA gene does not affect its rate of transcription by Polymerase III.

EXAMPLE 3 Inhibition of Viral Replication

The development and development of antisense tools are carried out, in the case of antiviral antisense, on permissive cell lines for the replication of the virus studied. For example, for anti-HIV antisense, the CEM or MOLT-4 lymphoblastoid lines, both good virus producers, are chosen.

The cells of the CEM line (free of mycoplasmas) were transfected with the various recombinant plasmids (pVV2 / VAI / anti-rev, pVV2 / VAI / anti-tat, pVV2 / VAI and control pVV2. The cell transfection techniques used depend on of the cell model studied.

For suspended cells, electroporation was preferably used (Gene Pulser (R) type device, Biorad, electric shocks are produced at 200V and 960 µF).

After transfection, the transduced cells are distributed in 96 independent culture wells and selected by the action of antibiotics geneticin 1.5 mg / ml (plasmid pVV2) or hygromycin 400 ug / ml (plasmid pHEBo). Maintaining the selection pressure remains necessary throughout the experiments. To date, using electroporation, the CEM line has been transfected with recombinant constructs of the plasmid pVV2 / anti-rev, pVV2 / anti-tat, pVV2 / VAI and pVV2.

Non-clonal, genetic-resistant cell populations from different culture wells are selected on the basis of good cell multiplication with a low mortality rate. The cell populations resulting from the transfection with “anti-sense” plasmids are numbered AS index X (AS: AntiSens, X n _° of the selection well).

Each population is infected with an infectious supernatant originating from cells chronically infected with the HIV virus (supernatant rich in particles), the strain used does not have a cytopathic effect on the cells used (strain LAV / BRU, JC Cherman, Marseille). The kinetics of viral replication show that, under usual conditions, more than 95% of EMFs produce viral particles 7 to 8 days after infection (Figure 4).

The measurement of viral replication is estimated by indirect immunofluorescence. Briefly, 5000 cells are fixed on glass slides and incubated for 30 min. in the presence of an anti-HIV serum from a seropositive patient diluted 1/40. The revelation is carried out through the use of a second antibody, specific for human immunoglobins, conjugated to fluorescein isothiocyanate (FITC).

Viral replication can also be measured by assaying the concentration of HIV-1 antigens present in the culture supernatant (assay carried out with the HIVAG-1 ^(R) kit from ABBOT). The kinetics of infection are carried out simultaneously on the cells transfected with the different plasmids and resulting from selection by geneticin.

Figure 4 presents the results obtained by infecting in a comparative manner the CEM line serving as a positive control, the CEM line resistant to geneticin (CEM.gén.r.) transfected with pVV2), 2 "anti-tat" populations (AS1 and AS2) and 2 "anti-rev" populations (AS3 and AS4). The measurement of viral replication was followed by observation and counting of the number of HIV positive cells detected by indirect immunofluorescence on different days after infection: The superposition of the curves "CEM" and "CEM gen.r." shows that the presence of geneticin in the medium does not affect the infection of CEM cells by the HIV virus. It appears that the 4 "antisense" populations tested show a significant delay in the start of the infection, the eclipse period is extended by 2 days for AS1 and up to 6 days in the case of AS3. In addition, a small percentage of cells are infected for certain lines (AS2, AS3, AS4) (25 to 60%) even late after infection, reflecting the resistance to viral infection of a large number of cells within these uncloned populations. This is not the case for the AS1 line which has more than 95% of positive cells 10 days after the start of the infection.

Figure 5 presents the results obtained during an experiment of the same type, but carried out with a larger panel of ASX populations and quantified by the measurement of the HIV-1 antigens present in the supernatants on different days after HIV infection. . Only the results obtained on day 24 are presented (these being representative of the results obtained on other days). In order to avoid the accumulation of viral particles and to distort the comparative measurements, the supernatants are collected 5 hours after the change of previous medium.

The populations "VAI / anti-tat" correspond to conditions AS1, AS2, AS6, AS7 and AS8, and the populations "VAI / anti-rev" correspond to conditions AS3, AS4, AS9, AS1 and AS12.

It appears that most “antisense” populations have a significant decrease in the amount of HIV-1 antigens measured in the supernatants, for 8 out of 10 lines (AS2, AS3, AS4, AS6, AS8, AS9, AS10 and AS12) , less than 20% of viral antigen production is measured compared to control, 2 populations reach 70% of the control level (AS1 and AS7).

The results obtained for populations AS 1,2,3 and AS4 according to 2 techniques for measuring viral replication: Immunofluorescence (Figure 4) and concentrations of antigens (Figure 5) are consistent. In Figures 4 and 5, the populations AS1, AS2, AS3 and AS4 are identical.

EXAMPLE 4 Measurement of the Expression Rate of VA-ARNI

It is possible to characterize the expression of antisense RNAs using Northen-blot techniques or hybridization in liquid medium. The RNAs are prepared according to the following protocol: 25.10 ⁶ are rinsed twice with saline buffer, the cells are lysed at 4 _° C by hypotonic shock (Tris 10 mM ph 7.4, NaCl 10 m ^M , MgCl ₂ , 3 mM ) and per share of NP 40 1%; after centrifugation (10,000 rpm, 10 minutes), the supernatant containing the cytoplasmic RNA is freed from residual proteins by 3 phenol-chloroform extractions and the RNA are concentrated with ethanol. 10 μg of RNA are analyzed by Northern blot and hybridized with a single strand probe specific for the 3 ′ region of the VA gene. The results obtained are presented in FIG. 8. The results presented here illustrate the fact that in vivo the constructs tested (here A5 _io , "VA / anti-rev") are expressed and that the transcription product obtained has indeed the expected size .

Example 5: Simultaneous use of several VA-chimeric genes: "Cocktails-antisense"

• Les ARN, issus de la transcription en système acellulaire du plasmide portant les deux gènes, ont été analysés par Northern-blot et révélés soit avec une sonde spécifique de la séquence rev, soit avec une sonde spécifique de la séquence tat. Les résultats obtenus sont présentés sur la figure 9.
  • - Le protocole de transcription acellulaire est celui déjà utilisé à l'exemple 2 et pour la figure 3 (ref 21 WU et al, et 22 Weil et al)
  • - L'analyse des ARN par Northern-blot est réalisé après purification des ARN (traitement par la DNase 1 puis la protéinase K et extraction au phénol-chloroforme), séparation sur gel d'agarose en milieu dénaturant, transfert sur membrane et révélation soit avec la sonde rev (piste a) soit avec la sonde tat (piste b).

A genetic construction was carried out comprising the succession of the two genes VA-anti rev and VA-anti tat, cloned respectively in the Cla1 and Hind III sites of the plasmid pVV2. Acellular transcription experiments, carried out with this construction, demonstrated that each of these genes expressed itself correctly:

• The RNAs, derived from the transcription in the acellular system of the plasmid carrying the two genes, were analyzed by Northern blot and revealed either with a probe specific for the rev sequence, or with a probe specific for the tat sequence. The results obtained are presented in FIG. 9.
  • - The acellular transcription protocol is that already used in Example 2 and for Figure 3 (ref 21 WU et al, and 22 Weil et al)
  • - Analysis of the RNA by Northern blot is carried out after purification of the RNA (treatment with DNase 1 then proteinase K and extraction with phenol-chloroform), separation on agarose gel in denaturing medium, transfer to membrane and revelation either with the rev probe (track a) or with the tat probe (track b).

Example 6: Vectorization of VA-antisense genes by modified murine retroviruses - Transfer to the progenitors of hematopoietic cells. 1) Type I retroviral vector:

The vector which is used is the plasmid pMV7 (P.T. Kirschmeier, GM. Housey, M.D. Jonhson, A.S. Perkins and I.B. Weinstein, 1988, DNA, Vol. 7, 3, 219-225). It is composed on the one hand of the sequences necessary for plasmid maintenance and on the other art of the two LTRs of the Moloney virus flanking the gene for resistance to neomycin (under the dependence of the TK proimotor). The Hind III, Clal and EcoRI cloning sites located between the 5 'LTR and the neomycin gene allow the insertion of exogenous sequences. The packaging cell is the DAMP cell (R. Mann, R.C. Mulligan and D. Baltimore, 1983, Cell, 33, 143-159). The genetic constructs were cloned at the Hind III site of pMV7 and transfected the recombinant plasmids obtained in the DAMP cell. DAMP cells that have become resistant to neomycin have been subcloned. The clones exhibiting a high infectious power, detected in the culture supernatants, were selected. These clones made it possible to infect lymphocytes of the CEM line (T4 auxiliary lymphocytes). The infection is carried out thanks to an overnight co-culture, between the DAMPs and the lymphocytes. Lymphocytes that have been infected have in turn become resistant to neomycin and can thus be selected. The RNAs from these lymphocytes were analyzed and the expression of the VA-anti rev gene (carried by the integrated proviral sequence) detected.

However, the possible interference between the activity of RNA Polymerase II which transcribes the entire viral genome from the 5 'LTR and the activity of RNA Polymerase III could, in certain cases, prove to be an element. unfavorable for the effectiveness of such a system.

2) Type II retroviral vectors:

The enhancer signals present in the U3 region of the 3 ′ LTR of the Moloney virus cloned in pMV7 were deleted and these signals were replaced by single cloning sites on this plasmid (these modifications being carried out using PCR technology). Antisense constructions are inserted at these sites. This modification has two advantages: it allows on the one hand to duplicate the cloned gene thanks to the game of reverse transcription (the U3 region present on each of the two LTRs comes from the U3 region located on the LTR in 3 ′ of the previous provirus) but above all, this no longer allows the RNA Polymerase II to transcribe the provirus integrated into the recipient cell. This avoids interference with RNA Polymerase III and ensures operational safety with regard to the always possible activation of adjacent genes.

• 1) le site unique Hind III a été délété (coupure, remplissage avec la DNA polymérase, religature).
• 2) la digestion complète du pMV7 par Cla I libère le gène de sélection (néomycine), la pârtie plasmidique résultante est ensuite traitée par l'enzyme Xho 1 de manière partielle. Seul le fragment contenant le LTR 5' est purifié sur gel d'agarose (5064 pb). De ce fait, le LTR 3' est perdu.
• 3) le LTR 3' est ensuite remplacé par un LTR 3' modifié pour lequel les régions enhancers situées dans la région U3 ont été supprimées et remplacées par un site Hind III (ceci est réalisé grâce à la technologie P.C.R.).
• 4) le gène antisens a été cloné dans le site Hind III nouvellement créé.
• 5) cette nouvelle construction rétrovirale peut être utilisée de manière habituelle :
  • - transfection dans des cellules d'encapsidation (cellules CRIP ref Danos and R.C. Mulligan, Proc. Natl. Acad. Sci., 1988, vol. 85, 6460-6446).
  • - récupération des surnageants avec estimation du titre infectieux.
  • - ces particules rétrovirales recombinantes seront utilisées pour transduire les cellules cibles.

More precisely, the vector Il was obtained as illustrated in FIG. 10:

• 1) the unique Hind III site has been deleted (cleavage, filling with DNA polymerase, religation).
• 2) the complete digestion of pMV7 by Cla I releases the selection gene (neomycin), the resulting plasmid tarnish is then partially treated with the enzyme Xho 1. Only the fragment containing the 5 'LTR is purified on agarose gel (5064 bp). As a result, the 3 'LTR is lost.
• 3) the 3 ′ LTR is then replaced by a modified 3 ′ LTR for which the enhancer regions located in the U3 region have been deleted and replaced by a Hind III site (this is achieved using PCR technology).
• 4) the antisense gene has been cloned into the newly created Hind III site.
• 5) this new retroviral construction can be used in the usual way:
  • - transfection into packaging cells (CRIP cells ref Danos and RC Mulligan, Proc. Natl. Acad. Sci., 1988, vol. 85, 6460-6446).
  • - recovery of supernatants with estimation of the infectious titer.
  • - these recombinant retroviral particles will be used to transduce the target cells.

In order to avoid obtaining wild recombinant particles of the Moloney virus, the DAMP packaging cell is changed in favor of the CRIP cell (O. Danos and RC Mulligan, Proc. Natl. Acad. Sci., 1988, 85, 6460-6446). The CRIP cell contains two helper retroviral genomes deleted from the 3 ′ LTR end of the packaging signal and mutated one in the Gag region and the other in the env region.

By complementation, the production of the proteins Gag, Pol and Env is ensured, but the probability of obtaining wild genomes obtained by recombination is reduced to zero. The set of improvements proposed and described here makes it possible to ensure the production of recombinant retroviral particles carrying one or more VA / antisense genes (pMV / AS) correctly transcribed by Polymerase III. This vector also offers optimum operational safety for the intended therapeutic applications.

EXAMPLE 7 Transient expression of VA-antisense genes

With a view to localized therapy in the short term, the transient expression of the VA-antisense genes has been verified in the cellular system. In transient expression, the VA-antisense gene is not integrated into the genome of the host cell and accumulates significantly in the nucleus where it is transcribed.

Adherent cells (line 293: kidney embryonic cells, human) were transfected with 20 g of the plasmid pVA anti rev. The technique used is transfection with calcium phosphate. This technique consists in putting in contact for 18 hours, the plasmid DNA precipitated by calcium phosphate and the cells by increasing the absorption of DNA on cell membranes and by limiting the action of cellular DNases on DNA. entering. The RNAs are prepared 48 hours after transfection, as described above, and analyzed by Northern blot with a probe specific for anti rev VA RNA. FIG. 11 shows a stronger transient expression of RNA (lane 293) in comparison with the RNA produced in cells which stably express the integrated VA RNA (lane CEM).

REFERENCES

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Claims (24)

1. Recombinant DNA vector comprising an RNA polymerase III transcription cassette, consisting of a viral gene transcribed by RNA polymerase III into which an oligonucleotide, DNA fragment, has been inserted between or outside boxes A and B constituting the promoter of said viral gene. 2. Vector according to claim 1, characterized in that the viral gene is a VA gene of an adenovirus, an EBER gene of an Epstein Barr virus or a viral gene transcribed by DNA polymerase III of a Herpes virus. 3. Vector according to claim 1 or 2, characterized in that the gene is the gene VA 1 or VA II of the Adenovirus. 4. Vector according to one of the preceding claims, characterized in that the vector is a plasmid, episomal replicative vector or a viral vector. 5. Vector according to one of the preceding claims, characterized in that the vector is an episomal vector carrying the origin of replication of the Epstein Barr virus (oriP), as well as the coding sequences for the protein EBNA 1. 6. Vector according to one of the preceding claims, characterized in that it comprises several identical or different viral genes transcribed by RNA polymerase III into which an identical or different oligonucleotide has been inserted outside of boxes A and B in each of said said viral genes. 7. Vector according to one of the preceding claims, characterized in that the oligonucleotide is inserted in the intermediate region located between the boxes A and B. 8. Vector according to one of the preceding claims, characterized in that the oligonucleotide comprises from 15 to 40 nucleotides, preferably 15 to 25. 9. Vector according to one of the preceding claims, characterized in that the viral gene is an inactivated Adenovirus gene, by deletion or mutation, as regards the inhibition of the action of interferon. 10. Vector according to one of the preceding claims, characterized in that the oligonucleotide is inserted inside the region responsible for the activity of inhibiting the action of interferon. 11. Vector according to one of claims 1 to 9 characterized in that an oligonucleotide is inserted in the region ranging from nucleotide 10694 to 10730, more particularly 10702 to 10728 or in place of this region of the VA-1 gene of l 'Adenovirus 2. 12. Vector according to one of the preceding claims, characterized in that the oligonucleotide is inserted into nucleotide 10711 of the VA 1 gene of the Adenovirus 2 represented in FIG. 1. 13. Vector according to one of claims 1 to 9 characterized in that an oligonucleotide is inserted into the VA-1 gene of Adenovirus 2 inside one of the following regions: a) nucleotides 10635 to 10639; b) nucleotides 10682 to 10688; c) nucleotides 10733 to 10736;
or in place of one of these regions, the latter being deleted. 14. Vector according to claim 13 characterized in that the oligonucleotide is inserted into the VA delta IV gene. 15. Vector according to one of the preceding claims, characterized in that it is a defective recombinant adenovirus. 16. RNA retroviral vector, characterized in that it comprises the RNA transcript of the transcription cassette according to one of claims 1 to 13. 17. Cells infected or transfected with a vector, according to one of claims 1 to 16. 18. A method of intracellular production of an RNA fragment in vitro or in vivo in which eukaryotic cells comprising RNA polymerase III are transfected or infected with a vector according to one of claims 1 to 16, comprising as oligonucleotides a DNA fragment corresponding to the reverse transcript of said RNA and in that the eukaryotic cells thus transfected or infected are cultured in an appropriate culture medium. 19. Method for blocking the expression of a gene in vivo using a vector, according to one of claims 1 to 16, in which said oligonucleotide is transcribed into an antisense or ribozymic RNA molecule which hybridizes and / or cuts the messenger RNA of said gene. 20. A method of treating cells ex vivo using a vector according to one of claims 1 to 16. 21. As a medicament, a vector according to one of claims 1 to 16 which can be used in particular as an antiviral, antitumor, antibiotic or antiparasitic agent, or in any pathology where a gene is abnormally expressed either by mutation or by deregulation. 22. As a medicament, according to claim 20, a vector according to one of claims 1 to 16, in which the oligonucleotide is transcribed into an "antisense" RNA molecule or a ribozymic RNA molecule blocking expression d '' a gene involved in a pathology by hybridizing and, if necessary, by cutting its messenger RNA of viral, bacterial or parasitic cell origin. 23. As a medicament according to claim 20 or 21, a vector constituted by a defective viral vector such as an Adenovirus or a defective retroviral vector such as a murine retrovirus. 24. As a medicament for the treatment of AIDS, according to one of claims 20 to 22, a vector according to one of claims 1 to 6, comprising as oligonucleotides antisense sequences in particular anti-rev or anti -state.

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